1. Field of the Invention
The present invention relates generally to carbohydrate biochemistry. More specifically, the present invention relates to starch biosynthesis and the enzyme(s) involved.
2. Description of the Related Art
Starch, the most significant carbohydrate reserve in plant storage tissues, comprises the glucose homopolymers amylose and amylopectin. Amylose consists of predominantly linear chains of .alpha.-(1.fwdarw.4)-linked glucose residues, whereas amylopectin is a highly branched glucan with a specific "clustered" distribution of .alpha.-(1.fwdarw.6) glycosidic bonds (i.e branch linkages) connecting linear chains French, 1984; Manners, 1989).
Despite the relatively simple chemical structure of amylopectin, very little is known about the enzymatic processes responsible for formation of the highly specific and complex branching patterns in this polysaccharide. Biosynthesis of amylose and amylopectin involves activities of four groups of enzymes, each of which comprises multiple isozymes. These enzymes are ADPG pyrophosphorylases (AGPase), starch synthases (SS), starch branching enzymes (SBE) and starch debranching enzymes (SDBE) (Preiss, 1991; Hannah et al., 1993; Martin and Smith, 1995; Nelson and Pan, 1995; Ball et al., 1996; Preiss and Sivak, 1996; Smith et al., 1996). These enzymatic steps can account for all chemical linkages in starch, however, the specific roles of individual isozymes in formation of specific branching patterns in amylopectin and determination of starch granule structure and properties remain unknown.
Analysis of maize mutants with abnormal endosperm phenotypes has contributed greatly to the understanding of starch synthesis (Shannon and Garwood, 1984; Nelson and Pan, 1995) and facilitated the identification of many genes coding for starch biosynthetic enzymes. Cloned genes whose products are thought to be involved directly in starch biosynthesis are waxy (wx), coding for the granule-bound starch synthase GBSSI (Shure et al., 1983; Klosgen et al., 1986), amylose extender (ae), coding for SBEIIb (Fisher et al., 1993; Stinard et al., 1993), shrunken2 (sh2) and brittle2 (bt2), coding for the large and small subunits of AGPase, respectively (Bae et al., 1990; Bhave et al., 1990), and sugaryl (su1), coding for the SDBE SU1 (James et al., 1995). The transposon-tagging strategy was used to determine that the abnormal endosperm phenotype of wx-, ae-, or su1- mutants results from primary defects in GBSSI, SBEIIb, or SU1, respectively, and this approach remains the most effective way to identify genes such as dull1 (du1), in which the primary defect can not be associated with a particular enzyme deficiency.
The du1- mutations define a gene with a very important function in starch synthesis, as indicated by extensive structural analyses of starch from du1- mutant endosperms, and by the effects of these mutations when combined with other genetic deficiencies in starch biosynthetic enzymes (Shannon and Garwood, 1984; Nelson and Pan, 1995). The reference mutation du1-Ref was first identified as a recessive modifier of su1-Ref and su1-amylaceous (su1-am) (Mangelsdorf, 1947). Mutations of du1, when homozygous in otherwise non-mutant backgrounds, result in mature kernels with a tarnished, glassy, and somewhat dull appearance referred to as the "dull phenotype". Expression of this phenotype, however, depends on the particular genetic background (Mangelsdorf, 1947; Davis et al., 1955). Total carbohydrate and starch content in mature du1-mutant kernels is slightly lower than normal (Creech, 1965; Creech and MeArdle, 1966). The apparent amylose content in starch from du1- mutants is slightly or greatly elevated compared to normal depending on the genetic background (Shannon and Garwood, 1984), although the properties of polysaccharides in the apparent amylose fraction are essentially not altered (Dvonch et al., 1951). Approximately 15% of the starch in du1- mutant endosperms is in a form known as "intermediate material", which is distinguished from amylose and amylopectin by the properties of its starch-iodine complex (Wang et al., 1993b). Analysis of combined amylopectin/intermediate material fractions indicated that starch from du1- mutants has the highest degree of branching among a wide variety of normal and mutant kernels analyzed (Inouchi et al., 1987; Wang et al., 1993a; Wang et al., 1993b). Starch granules from du1- mutants seem to have normal structural and physical properties, although some abnormally shaped granules are found in the mutant endosperm (Shannon and Garwood, 1984).
Despite these subtle effects exerted by the single mutation, du1-alleles when combined with other mutations affecting starch synthesis result in a broad range of more severe alterations (Shannon and Garwood, 1984; Nelson and Pan, 1995). Mutations of du1 have been examined in combination with wx-, ae-, su1-, and sugary2 (su2-) mutations, and in all instances the double mutant kernels contained more soluble sugars and less total starch than when any of the mutations was present alone. In many instances the double mutants also produce polysaccharide forms that are distinct from the starch found in any single mutant kernels. These pleiotropic effects indicate the product of Du1 affects many aspects of starch biosynthesis in maize endosperm, however, without knowing the identity of this protein it is difficult to assess its specific functions.
Consistent with the pleiotropic genetic effects, du1-mutations cause reduced activity in endosperm of two seemingly unrelated starch biosynthetic enzymes, the starch synthase SSII and the branching enzyme SBEIIa (Boyer and Preiss, 1981). SSII is one of two enzymatically distinct starch synthase activities identified in the soluble fraction of maize endosperm; in vitro activity of SSII requires an exogenous glucan primer, and its molecular weight was determined in different studies as either 95 kD or 180 kD (Boyer and Preiss, 1981; Mu et al., 1994). Similarly, SBEIIa is one of the three known SBE isozymes in endosperm cells (Boyer and Preiss, 1978b; Fisher et al., 1993; Fisher et al., 1995; Gao et al., 1997). Several possibilities exist to explain the dual biochemical effects of du1- mutations. Du1 may code for a protein regulating the expression or activity of both SSII and SBEIIa. Alternatively, Du1 may code for either of these two enzymes, and the deficiency in one enzyme might also affect the second enzyme because of a direct or substrate-mediated physical interaction.
DU1 codes for a starch synthase, as indicated by the extensive similarity of its deduced amino acid sequence to potato SSIII, and by the substantial similarity between the C-terminal residues of DU1 and a large group of phylogenetically diverse starch- and glycogen synthases. Particularly striking are two regions that together comprise more than half of the deduced DU1 sequence of 1,674 residues, which share very high similarity of 51% and 73%, respectively, with the corresponding regions of the potato SSIII sequence. Within a stretch of 450 amino acids at the C-terminus of DU1 nearly 30% of the best aligned residues are identical in comparisons to a wide variety of starch- and glycogen synthases, suggesting the location of a domain within DU1 that provides .alpha.-1,4-glycosyltransferase acitivity.
The starch synthase coded for by Du1 is the soluble isozyme identified biochemically as SSII (Ozbun et al., 1971; Boyer and Preiss, 1981). The deduced molecular weight of Du1 including a potential transit peptide, 188 kD, matches closely with that of 180 kD reported for mature SSII lacking a transit peptide (Mu et al., 1994). The size difference of approximate 8 kD may be due to the transit peptide present in the deduced DU1 sequence. The tissue specific expression pattern of the Du1 mRNA also matches the expression pattern of SSII. Du1 transcripts were undetectable in leaves either by RNA gel blot or RT-PCR analyses, corresponding with that fact no detectable SSII activity was present in leaf extracts (Dang and Boyer, 1988). Moreover, the activity of SSII, along with that of SBEIIa, was greatly reduced in du1-mutant endosperm (Boyer and Preiss, 1981). Therefore, it appears that the maize du1 locus codes for the soluble starch synthase SSII, the counterpart of potato SSIII.
This characterization of Du1 implies that the phenotypic effects of du1- mutations, including changes in starch structure, deficiencies of two starch biosynthetic enzymes, and genetic interactions with ae-, su1-, su2-, and wx- mutations, all result either directly or indirectly from alteration of SSII. The reduction of SBEIIa activity in du1-mutant endosperm could result from the SSII deficiency owing to physical interaction between the two enzymes. A direct physical association of SSII and SBEIIa is implied by the observation that peak activities of both SSII and SBEIIa always coincide in the same DEAE-cellulose column fractions (Boyer and Preiss, 1978a; Boyer and Preiss, 1981; Dang and Boyer, 1988). Thus, SSII and SBEIIa may function together in vivo in the form of single multi-enzyme complex. Loss of the intact enzyme complex owing to reduction of SSII in du1-mutant endosperm may result in abnormally rapid proteolytic turnover of SBEIIa, or prevent accumulation of the enzyme by some other mechanism. Alternatively, expression of the Sbe2a gene in du1 -mutant endosperm may be inhibited as a more indirect consequence of the deficiency in SSII, for example through reduction of a transcriptional inducer or elevation of a repressor. Although the du1-Ref mutation does indirectly affect expression of other starch biosynthetic genes (Giroux et al., 1994), it actually caused increased gene expression rather than the reduction observed for SBEIIa. Furthermore, considering that large glucose polymers are expected to be the substrate and product of DUI, down-regulation of Sbe2b expression by a transcriptional mechanism seems unlikely. Thus, the former hypothesis may explain the deficiency of SBEIIa in du1-mutant endosperm.
The broad impact of the combination of du1- mutations with various su1-alleles on kernel phenotype and starch synthesis (Cameron, 1947; Shannon and Garwood, 1984) could be explained by the SU1 SDBE also interacting closely with SSII in vivo, perhaps in the same enzyme complex with SBEIIa. This proposed association of SBEIIa and SU1 in a multi-enzyme complex is consistent with the proposed simultaneous branching and debranching actions during amylopectin synthesis by SBE and SDBE (James et al., 1995; Nelson and Pan, 1995; Ball et al., 1996).
Thus, the prior art is deficient in understanding the complex association of enzymes involved in starch synthesis and in cloning genes corresponding to these enzymes. The present invention fulfills this long-standing need and desire in the art.